Gustducin materials and methods

ABSTRACT

A novel taste cell specific guanine nucleotide binding protein, gustducin, is disclosed as well as polynucleotide sequences encoding the α subunit of gustducin. Also disclosed are methods of modifying taste involving agents that inhibit or activate the gustducin α subunit, methods for identifying such taste modifying agents and various taste modifying agents.

This is a continuation of U.S. application Ser. No. 08/407,804, filedMar. 20, 1995 now U.S. Pat. No. 5,817,759, which is acontinuation-in-part of U.S. application Ser. No. 07/868,353 filed Apr.9, 1992 now U.S. Pat. No. 5,688,662.

FIELD OF THE INVENTION

The present invention relates, in general, to materials and methodsrelevant to taste transduction. More particularly, the invention relatesto a heretofore unknown taste cell specific guanine nucleotide bindingprotein, gustducin, and to polynucleotide sequences encoding the αsubunit of gustducin. The invention also relates to methods of modifyingtaste that involve agents which inhibit or activate the gustducin αsubunit, to methods for identifying such taste, modifying agents and tothe taste modifying agents.

BACKGROUND

Vertebrate taste transduction is mediated by specialized neuroepithelialcells, referred to as taste receptor cells, organized into groups offorty to one hundred cells which form taste buds. Taste buds are ovoidstructures, the vast majority of which are embedded within theepithelium of the tongue. Taste transduction is initiated at the apicalportion of a taste bud at the taste pore where microvilli of the tastereceptor cells make contact with the outside environment. Various tastestimulants (tastants) cause either depolarization (i.e., a reduction inmembrane potential) or hyperpolarization (i.e., an increase in membranepotential) of taste cells and regulate neurotransmitter release from thecells at chemical synapses with afferent nerve fibers. The primarygustatory sensory fibers which receive the chemical signals enter thebase of each taste bud. Lateral connections between taste cells in thesame bud may also modulate the signals transmitted to the afferent nervefibers.

There are four basic taste modalities typified by four distinct groupsof taste stimuli: salty, sour, sweet, and bitter. Different tastemodalities appear to function by different mechanisms. For example,salty taste appears to be mediated by sodium ion flux through apicalsodium channels [see Heck et al. Science, 223, 403-405 (1984) andSchiffman et al., Proc. Natl. Acad. Sci USA, 80, 6136-6140 (1983)] andsour taste seems to be mediated via hydrogen ion blockade of potassiumor sodium channels [see Kinnamon et al., J. Gen. Physiol., 91, 351-371(1988) and Kinnamon et al., Proc. Natl. Acad. Sci. USA, 85, 7023-7027(1988)].

Of particlar interest to the background of the present invention areguanine nucleotide binding proteins (G proteins) which have beenspecifically implicated in the transduction of sweet and bitter tastesand may also be involved in the regulation of the ion charnels involvedin transduction of salty and sour tastes. See, for example, the recentreviews on G proteins: Birnbaumer, Ann. Rev. Pharmacol. Toxicol., 30,675-705 (1990) and Simon et al., Science, 252, 802-808 (1991). Briefly,G proteins are heterotrimeric proteins (each having an α, β, and γsubunit) which mediate signal transduction in olfactory, visual,hormonal and neurotransmitter systems. G proteins couple cell surfacereceptors to cellular effector enzymes (e.g., phosphodiesterases andadenylate cyclase) and thereby transduce an extracellular signal into anintracellular second messenger (e.g., cAMP, cGMP, IP₃). The α subunit ofa G protein confers most of the specificity of interaction between itsreceptor and its effectors in the signal transduction process, while βand γ subunits appear to be shared among different G proteins. Some Gproteins are ubiquitously expressed (e.g., G_(s) and G_(i)), but othersthat are known to be involved in sensory transduction have been foundonly in specialized sensory cells. For example, the transducins (G_(t))transduce photoexcitation in retinal rod and cone cells [see Lerea etal., Science, 224, 77-80 (1986)], and G_(olf) transduces olfactorystimulation in neurons of the olfactory epithelium [see Jones et al.,Science, 244, 790-795 (1989)]. The ubiquitously expressed G proteins mayalso be involved in sensory transduction.

While no direct evidence for the existence of a gustatory specific Gprotein has been previously reported, experimental data suggesting thatG proteins are involved in the taste transduction pathway is describedin several publications, including, for example, the reviews of Kinnamonet al., TINS, 11(11), 491-496 (1988); Avenet et al., J. Membrane Biol.,112, 1-8 (1989); and Roper, Ann. Rev. Neurosci., 12, 329-353 (1989).

Avenet et al., Nature, 331, 351-354 (1988) and Tonosaki et al., Nature,331, 354-356 (1988) report that external application or microinjectionof cAMP inactivates potassium channels in vertebrate taste cells andleads to depolarization of these cells. Kurihara et al., Biophys. Res.Comm., 48, 30-34 (1972) and Price et al., Nature, 241, 54-55 (1973)describe high levels of adenylyl cyclase and cAMP phosphodiesterase intaste tissue.

In Striem et al., Biochem. J., 260, 121-126 (1989), sweet compounds areproposed to cause a GTP-dependent generation of cAMP in rat tonguemembranes. These results suggest a transduction pathway in which tastantinteraction with a sweet receptor leads to taste cell depolarization viaa G protein mediated rise in cAMP. Akabas et al., Science, 242,1047-1050 (1988) reports that bitter compounds such as denatonium leadto Ca²⁺ release from internal stores. The release may be a result of Gprotein-mediated generation of inositol trisphosphate (IP₃). Thus,bitter taste may also be transduced via a G protein.

Over the past decade substantial efforts have been directed to thedevelopment of various agents that interact with taste receptors tomimic or block natural taste stimulants. See, Robert H. Cagan, Ed.,Neural Mechanisms in Taste, Chapter 4, CRC Press, Inc., Boca Raton, Fla.(1989). Examples of agents that have been developed to mimic sweettastes are saccharin (an anhydride of o-sulfimide benzoic acid) andmonellin (a protein) and the thaumatins (also proteins). Thaumatins havebeen utilized as additives in food, cigarette tips, medicines andtoothpaste [Higginbotham et al, pp. 91-111 in The Quality of Foods andBeverages, Academic Press (1981)]. Many taste-mimicking ortaste-blocking agents developed to date are not suitable as foodadditives, however, because either they are not economical or are highin calories, or because they are carcinogenic. Development of new agentsthat mimic or block the four basic tastes has been limited by a lack ofknowledge of the taste cell proteins responsible for transducing thetaste modalities. There thus continues to exist a need in the art fornew products and methods that are involved in or affect tastetransduction.

SUMMARY OF THE INVENTION

The present invention provides products and methods that are involved inor that affect taste transduction. In one of its aspects, the presentinvention provides purified and isolated polynucleotide sequences (e.g.,DNA sequences and RNA transcripts thereof) encoding the α subunit of anovel taste receptor cell specific G protein, gustducin, or fragmentsand variants of the α subunit that possess at least oneligand/antiligand binding activity or immunological property specific togustducin. Preferred polynucleotide sequences of the invention includegenomic and cDNA sequences as well as wholly or partially synthesizedDNA sequences, and biological replicas thereof. Biologically activevectors comprising the polynucleotide sequences are also contemplated.

The scientific value of the information contributed through thedisclosures of the DNA and amino acid sequences of the present inventionis manifest. For example, knowledge of the sequence of a cDNA encodingthe gustducin α subunit makes possible the isolation by DNA/DNAhybridization of genomic DNA sequences that encode the subunit and thatspecify α subunit-specific expression regulating sequences such aspromoters, operators and the like. DNA/DNA hybridization proceduresutilizing the DNA sequences of the present invention also allow theisolation of DNAs encoding heterologous species proteins homologous tothe rat gustducin α subunit specifically illustrated herein, such ashuman species gustducin α subunit protein.

According to another aspect of the invention, host cells, especiallyunicellular eucaryotic and procaryotic cells, are stably transformed ortransfected with the polynucleotide sequences of the invention in amanner allowing the expression of gustducin α subunit polypeptides inthe cells. Host cells expressing gustducin α subunit polypeptideproducts, when grown in a suitable culture medium, are particularlyuseful for the large scale production of gustducin α subunitpolypeptides, fragments and variants; thereby enabling the isolation ofthe desired polypeptide products from the cells or from the medium inwhich the cells are grown.

The novel gustducin α subunits, fragments and variants of the inventionmay be obtained as isolates from natural taste cell sources, but arepreferably produced by recombinant procedures involving the host cellsof the invention. The products may be obtained in fully or partiallyglycosylated, partially or wholly de-glycosylated or non-glycosylatedforms, depending on the host cell selected or recombinant productionand/or post-isolation processing. The products may be obtained in fullyor partially myristoylated, partially or wholly de-myristoylated ornon-myristoylated forms, depending on the host cell selected orrecombinant production and/or post-isolation processing.

Gustducin α subunit variants according to the invention may -comprisepolypeptide analogs wherein one or more of the specified amino acids isdeleted or replaced or wherein one or more nonspecified amino acids areadded: (1) without loss, and preferably with enhancement, of one or moreof the biological activities or immunological characteristics specificfor gustducin; or (2) with specific disablement of a particularligand/antiligand binding function.

Also contemplated by the present invention are antibody substances(e.g., monoclonal and polyclonal antibodies, chimeric and humanizedantibodies, and antibody domains including Fab, Fab', F(ab')₂ and singlechain domains, and Fv or single variable domains) which are specific forthe gustducin α subunit.

Antibody substances can be developed using isolated natural orrecombinant gustducin α subunit polypeptide products or host cellsexpressing such products on their surfaces. The antibody substances maybe utilized for purifying polypeptides of the invention and for blockingor inhibiting ligand/antiligand binding activities of gustducin.

Yet another aspect of the present invention relates the observation thatgustducin α subunit polypeptides (and by virture of their sequencehomology to gustducin, rod or cone transducing α subunit polypeptides)are particularly suited for use in methods for identifying tastemodifying agents. Methods of identifying taste modifying agentsaccording to the invention generally involve testing an agent for thecapability to mimic or inhibit the interaction of gustducin α subunitwith a sensory receptor or for the capability to mimic or inhibit theinteraction of gustducin α subunit with an effector enzyme.

A first preferred method for identifying a taste modifying agentcomprises the steps of incubating phospholipid vesicles having gustducinα subunit or transducin α subunit and G protein β and γ subunitsassociated in biologically active form with an agent and withradioactively labeled GTPγS, and determining the rate of GTPγS bindingby the α subunit in comparision to a standard rate. An increase in therate of binding indicates that the agent is a taste stimulator and adecrease in the rate of binding indicates that the agent is a tasteinhibitor.

A second preferred method for identifying a taste modifying agentincludes the steps of incubating phospholipid vesicles having gustducinα subunit or transducin α subunit and G protein β and γ subunitsassociated in biologically active form with a particular agent andradioactively labeled GTP, and determining the rate of conversion of GTPto GDP by the α subunit in comparison to a standard rate. An increase inthe rate of conversion indicates that the agent is a taste stimulatorand a decrease in the rate of conversion indicates that the agent is ataste inhibitor.

A third preferred method for identifying a taste modifying agentcomprises the steps of incubating activated gustducin α subunit oractivated transducin α subunit with an agent and a phosphodiesterase,and measuring phosphodiesterase activation by the α subunit incomparison to a standard. An increase in phophodiesterase activityindicates the agent is a taste stimulator and a decrease inphosphodiesterase activity indicates that the agent is a tasteinhibitor.

A fourth preferred method for identifying a taste modifying agentincludes the steps of incubating washed disk membranes (e.g., frombovine retina) with gustducin α subunit or transducin α subunitassociated with G protein if and γ subunits in biologically active formwith a particular agent, subjecting the membranes to photolyzingconditions (i.e., 532 nm light), and determining absorption ofphotolytic reaction products at 380 nm in comparison to a standard. Anincrease in absorption at 380 nm indicates that the agent is a tastestimulator and a decrease in absorption at 380 nm indicates that theagent is a taste inhibitor.

Taste modifying agents may, for example, comprise a peptide possessingat least one ligand/antiligand binding activity specific to the αsubunit of gustducin. Amino acid sequences of presently preferred tastemodifying peptides are set out in SEQ ID NOs: 1-10, wherein SEQ ID NOs:1-3 correspond to the carboxyl terminal region of rat gustducin αsubunit, SEQ ID NO: 4 corresponds to the amino terminal portion ofbovine transducin, SEQ ID NOs: 5-7 correspond to the carboxyl terminalportion of bovine transducin, SEQ ID NOs:

8-10 correspond to loop peptides of bovine rhodopsin, SEQ ID NO: 11corresponds to amino acids 297-318 of rat gustducin, SEQ ID NO: 12 10corresponds to amino acids 304-318 of rat gustducin, SEQ ID NO: 13corresponds to amino acids 57-69 of rat gustducin, SEQ ID NO: 14corresponds to amino acids 293-314 of bovine rod transducin, SEQ ID NO:15 corresponds to amino acids 300-314 of bovine rod transducin, SEQ IDNO: 16 corresponds to amino acids 53-65 of bovine rod transducin, SEQ IDNO: 17 corresponds to amino acids 297-318 of bovine cone transducin, SEQID NO: 18 corresponds to amino acids 304-318 of bovine cone transducin,and SEQ ID NO: 19 corresponds to amino acids 57-69 of bovine transducin.Taste modifying peptides may be acetylated at the amino terminus oramidated at the carboxyl terminus.

Other peptide ligands/antiligands of the gustducin α subunit may beidentified by contacting gustducin α subunits with peptides andisolating the peptides which bind to the subunits. Appropriate peptidedisplay libraries or phage epitope libraries which may be utilized insuch methods are described in Scott et al., Science, 249, 386-390(1990); Lam et al., Nature, 354, 82-84 (1991);

and Houghton et al., Nature, 354, 84-86 (1991).

According to another aspect of the present invention, taste modifyingagents such as peptides having a ligand/antiligand binding activity ofgustducin α subunit or an antibody substance specific for gustducin αsubunit are delivered to taste receptor cells to modify taste (e.g.,mimic or inhibit sweet and/or bitter tastes).

Numerous aspects and advantages of the present invention will beapparent upon consideration of the illustrative examples anddescriptions in the following detailed description thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B is an alignment of amino-acid sequences of the α subunits ofrat gustducin (SEQ ID NO: 21), bovine cone transducin (cone) (SEQ ID NO:22), bovine rod transducin (rod) (SEQ ID NO: 23) and a consensussequence (SEQ ID NO: 24) derived from the alignment of the three αsubunits, wherein captial letters in the consensus sequence indicatethat all three subunits have the same amino acid at that position,lower-case letters indicate two of the three proteins have the sameamino acid at that position, and dots indicate all three subunits have adifferent amino acid at that position.

DETAILED DESCRIPTION

The present invention is illustrated by the following examples whereinExample 1 describes the cloning cDNA sequences encoding the α subunit ofrat species gustducin; Example 2 presents characterizations of thegustducin α subunit cDNA; Example 3 describes experiments relating tothe expression of the α subunit of gustducin in E. coli; Example 4presents the results of Northern blot, primer extension and RNaseprotection assays for the expression of gustducin α subunit mRNA invarious tissues; Example 5 describes methods for identifying tastemodifying agents having the capability to affect interactions betweenthe gustducin α subunit and taste receptors or effectors and alsodescribes methods for utilizing such taste modifying agents to modifytaste by mimicking or inhibiting sweet, bitter, salty or sour tastes;and Example 6 describes the generation of gustducin α subunit specificpolyclonal antibodies.

EXAMPLE 1

A cDNA clone encoding a heretofore unknown taste cell specific G proteinwas isolated by PCR from a taste cell enriched cDNA library. Taste budswere estimated to comprise 10-30% of the total mass of taste tissueharvested to make the library. In contrast, taste buds represent lessthan 1% of the total lingual epithelium. A control cDNA library was madefrom lingual epithelium devoid of taste buds.

Construction of cDNA Libraries

The circumvallate and foliate papillae from ninety Sprague-Dawley ratswere harvested by the method described in Spielman et al., Chem. Senses,14, 841-846 (1989) and immediately frozen in 100% ETOH at -70° C. Anequivalent amount of non-taste lingual epithelium (devoid of taste buds)was likewise harvested. Poly A+ mRNA was isolated from taste andnon-taste lingual tissue using a Quick Prep kit (Pharmacia, Upsala,Sweden). 7.9 μg of mRNA was recovered from the taste tissue and 2.4 μgof mRNA was recovered from the control non-taste lingual tissue. TheSuperscript kit (BRL, Bethesda, Md.), which utilizes the pSPORT vector,was used to make two cDNA libraries from 1 μg of taste and 1 μg ofnon-taste lingual mRNA. The taste library contained 2.6×10⁶ independentclones (average insert size of 1.1 kb). The non-taste library contained4.8×10⁶ independent clones (average insert size of 1.0 kb).

Design and Sythesis of PCR Primers

Six degenerate oligonucleotide primer sets were made that correspondedto regions of amino acids highly conserved among previously described Gprotein α subunits including α_(s), α_(olf), α_(i-1),3, α_(i-2), α_(o),α_(z), α_(q), α_(t-rod), and α_(t-cone) subunits. The amino acidsequences of the conserved regions and the DNA sequences (in IUPACnomenclature) of the corresponding degenerate primer sets, which weresynthsized on an Applied Biosystems DNA synthesizer, are set out below.Oligonucleotides corresponding to 3' primers (sets 2, 4, and 6) weresynthesized in the antisense orientation. Underlined sequences at theend of each oligonucleotide contain a restriction endonuclease site(BamHl for oligonucleotides used as 5' primers and EcoRloligonucleotides used as 3' primers) to facilitate cloning. Thenucleotide number (Nuc. #) in parentheses refers to the gustducin αsubunit nucleotide location now known to correspond to the first aminoacid of the primer.

Set 1

    KWIHCF (Nuc. 741)                                          (SEQ ID NO: 25)

    5' CGGATCCAARTGGATHCAYTGYTT 3'                             (SEQ ID NO: 26)

Set 2

    FLNKKD (Nuc. 912)                                          (SEQ ID NO: 27)

    5' GGAATTCRTCYTTYTTRTTNAGRAA 3'                            (SEQ ID NO: 28) and

    5' GGAATTCRTCYTTYTTRTTYAARAA 3'                            (SEQ ID NO: 29)

Set 3

    DVGGQR (Nuc. 711)                                          (SEQ ID NO: 30)

    5' GTCTAGAGAYGTNGGNGGNCARMG 3'                             (SEQ ID NO: 31)

Set 4

    VFDAVTD (Nuc. 1116)                                        (SEQ ID NO: 32)

    5' CCGAATTCTCNGTNACNGCRTCRAANAC 3'                         (SEQ ID NO: 33)

Set 5

    TIVKQM (Nuc. 255)                                          (SEQ ID NO: 34)

    5' CCGAATTCACNATNGTNAARCARATG 3'                           (SEQ ID NO: 35)

Set 6

    FLNKQD (Nuc. 912)                                          (SEQ ID NO: 36)

    5' CCGAATTCRTCYTGYTTRTTNARRAA 3'                           (SEQ ID NO: 37)

Primer sets 1, 2 and 3 were previously described in Strathmann et al.,Proc. Natl. Acad. Sci. USA, 86, 7407-7409 (1990). The two degenerateoligonucleotides comprising set 2 were always used together in equimolaramounts.

Cloning of cDNA Encoding the Gustducin α Subunit by PCR

DNA from the taste cell library was used as a substrate for PCR usingseveral pairwise combinations of two of the foregoing degenerate primersets: 1 and 2, 2 and 3, and 5 and 6. PCR samples contained 250 pmol ofeach primer, 20 ng of taste cell library cDNA, and 1 unit pyrostase(Molecular Genetic Resources, Tampa, Fla.) in a 50 μl reaction volume.The PCR program was: 94° for 1 minute, 37° to 72° with a rise time of 1°per 4 seconds, then 72° for 3 minutes for three cycles; followed by 94°for 1 minute, then 43° for 2 minutes, and finally 72° for 3 minutes fora total of 35 additional cycles. The PCR products were digested withBamHI and EcoRI, and electrophoresed in a 1% agarose gel. Bands ofexpected size were excised, purified, cloned into the pBluescript vector(Stratagene, La Jolla, Calif.), and transformed into E. coli. Individualcolonies were picked, and the DNA isolated therefrom was sequenced.

Partial clones were categorized according to cr subtype specificitybased on their deduced amino acid sequence. Eight different types of αsubunit clones were isolated. Seven of the α subunit types (α_(s), twotypes of α_(i), two types of α_(q), and two types of α_(t)) had beenpreviously identified and are expressed in tissues other than lingualepithelium. The eighth type of clone (generated in PCR reaction usingprimer sets 1 and 2, and 5 and 6) was a novel G protein α subunit clone.This gustatory clone was one of the most frequent isolates, suggestingthat it is present in relatively high abundance in the taste tissue cDNAlibrary.

To determine the complete sequence of the gustatory α subunit clone bothfurther PCR reactions and colony hybridization to the taste cell cDNAlibrary using PCR products as probes were performed.

PCR reactions were performed as described above using the α subunitspecific primer set out below (which was synthesized in the antisenseorientation and has a BamHI site at its 5' end) and degenerate primerset 4.

    HLFNSIC (Nuc. 855)                                         (SEQ ID NO: 38)

    5' CCGGATCCGCACCTGTTCAACAGCATCT 3'                         (SEQ ID NO: 39)

The PCR fragments generated were cloned and sequenced as describedabove.

Nested PCR reactions using the α subunit specific primers indicatedbelow were performed to obtain gustatory α subunit 5' sequences.

    KYFATTS (Nuc. 882)                                         (SEQ ID NO: 40)

    5' CCGGATCCGAGGTGGTTGCAAAATACTT 3'                         (SEQ ID NO: 41)

    LAEIIKR (Nuc. 480)                                         (SEQ ID NO: 42)

    5' CGGATCCGACGTTTAATTATTTCAGCCAA 3'                        (SEQ ID NO: 43)

The primer set out in SEQ ID NO: 41 and a T7 sequencing primer (BRL),which corresponds to the T7 promoter region of the pSPORT vectorcontaining the taste cell library, were used as primers in a first PCRreaction. Next, the PCR fragments generated were reamplified using theprimer set out in SEQ ID NO: 43 and the T7 sequencing primer (BRL). Thereamplfied fragments were then cloned and sequenced as described above.

A PCR fragment amplified using primer set 5 and the primer set out inSEQ ID NO: 41 was used as a probe for colony hybridizations to the rattaste cell cDNA library to obtain/confirm the gustatory α subunitsequence. Clones designated T95, T93, T85 and T77 were isolated andsequenced.

A composite gustatory α subunit clone was assembled in the plasmidvector pSPORT (BRL) and the resulting plasmid was designatedpSPORT-gustducin. Clone T95 (comprising the pSPORT vector and gustatoryα subunit sequences) was digested with NsiI (an endonuclease which doesnot cut within the pSPORT vector, but cuts at two sites within the αsubunit DNA at nucleotides 354 and 886) to yield two fragments. Thelarger fragment (˜5250 bp containing pSPORT vector sequences and most ofthe gustatory α subunit sequences) was recovered after being isolatedaway from the smaller fragment (˜400 bp). A fragment containing theremaining gustatory α subunit sequences was derived from PCRamplification of the taste cell cDNA library with primer set 5 and thegustatory α subunit specific primer set out in SEQ ID NO: 41. The PCRproduct generated was digested with NsiI resulting in a 532 bp fragment.The 532 bp fragment was then ligated to the large fragment isolated fromclone T95 to generate a composite α subunit clone in the vector pSPORT.The 5' end of the gustatory α subunit cDNA is coupled to sequencesderived from a SalI/MluI adaptor used to make the original cDNA libraryin the vector pSPORT (vector . . . 5' TCGACCCACGCGTCCG 3'/5'gustducin)[i.e., vector . . . (SEQ ID NO: 44)/5'gustducin). The 3' end of thegustducin cDNA is coupled to the T-tailed NotI primer-adapter used inthe original pSPORT library construction (gustducin 3'/5' GGGCGGCCGC 3'. . . vector) [i.e., gustducin 3'/(SEQ ID NO: 45) . . . vector].

The DNA and deduced amino acid sequences of the composite gustatory αsubunit clone are respectively set out in SEQ ID NOs: 20 and 21. The Ssequences were published in McLaughlin et al., Nature, 357, 563-569(1992). The gustatory α subunit sequence consists of 1703 bp of DNA witha single long open reading frame sufficient to encode a protein of 354amino acids. It contains potential sites for pertussis toxin (C₃₅₁) andcholera toxin (R₁₇₈) mediated ribosylation.

Transducin in Taste Cells

Interestingly, transducin α subunit cDNAs (both rod and cone) wereisolated by PCR amplification of the taste cell library. Furthermore,transducin α subunit mRNA was shown to be present-in taste buds by RNaseprotection assays and by in situ hybridization. This was the firstdemonstration of the presence of transducin in a tissue other than thephotoreceptor cells of the retina. Transducin may therefore participatein taste transduction as well as visual transduction.

EXAMPLE 2

Comparison of the Sequence of the Gustatory

α Subunit Clone with Known G Protein α Subunits

The Tfasta and Fasta programs of the Wisconsin GCG software packagedescribed in Devereaux et al., Nucl. Acids Res., 12, 387-395 (1984),were used to search GenBank for DNA and amino acid sequences related tothe α subunit of rat gustatory protein. The search revealed that the αsubunit is a member of the α_(i) superfamily and is most closely relatedto the bovine rod and bovine cone transducins. Due to its closerelationship to the transducins and its presumptive role in tastetransduction, the gustatory G protein was named gustducin. At the aminoacid level, the α subunit of rat gustducin is 80% identical and 90%similar to the α subunit of bovine rod transducin, and is 79% identicaland 90% similar to the α subunit of bovine cone transducin. Incomparison, bovine rod a transducin is 81% identical and 90% similar tobovine cone a transducin. Since the rat transducin α subunit DNAsequences have not been determined, a comparison of rat gustducinαsubunit to rat transducin α subunits could not be made. However, amongmammals, a 1 to 3% difference in amino acid identity is typical among αisotypes, suggesting that the α subunits of gustducin and thetransducins comprise a subfamily of closely related proteins. Incontrast, gustducin α subunit is only about 67% identical to the α_(i)subunits, and only 46% identical to α_(s) subunits (similar levels ofhomology exist between the transducins and α_(i) or α_(s)).

An alignment of gustducin α subunit with the α subunits of bovine rodand cone transducin produced iteratively by the BestFit routine of theWisconsin GCG software package (Devereaux et al., supra) shows that thegeneral structure of all three α subunits is highly conserved (see FIGS.1A-1B). The amino terminal 60 amino acids and the carboxyl terminal 60amino acids of all three proteins are highly conserved, while thecarboxyl terminal 38 amino acids are identical. This carboxyl terminalidentity is of particular importance because it encompasses the sitethat has been implicated in G protein/receptor interactions. Moreover,the region from Q₁₃₇ through F₃₅ is extremely similar for all threesubunits; each α subunit has only 14 or 15 differences from theconsensus sequence in this region. This region contains most of thesites implicated in guanine nucleotide binding. Amino acids G₄₂, R₁₉₇and Q₂₀₄ regulate GTPase activity and are present in all three proteins.These comparisons suggest that the guanine nucleotide binding propertiesand GTPase activities of these three α subunits are likely to be quitesimilar. All three α subunits contain a potential N-myristoylation siteat their terminus which, if utilized, may anchor these α subunits to theinner face of the plasma membrane. Most differences among the threeproteins are clustered in the region from V₉₆ to S₁₀₉ of gustducin,which is a highly variable region of G protein α subunits.

Gustducin α Subunit is a Single Copy Gene

Although the α subunit of gustducin is closely related to the transducinα subunits, it differs at the amino acid level at several positionsscattered throughout its sequence. This suggests that α gustducin istranscribed from a gene distinct from the transducins. Southern blotanalysis with gustducin α subunit probes vs. transducin α subunit probesconfirmed that gustducin is a single copy gene with a distinctrestriction endonuclease digestion pattern.

Splice Variants of Gustducin α Subunit

In screening the taste enriched cDNA library two apparent splicingvariants of the α subunit of gustducin were found. One type of clone(T95) contained the entire coding region of the gustducin α subunit, buthad an in frame deletion of 135 bp. When this cDNA sequence is alignedwith the genomic sequence of the α subunit of murine transducin, thedeletion corresponds to the precise removal of the sixth exon. Thegeneral exon-intron organization of G protein α subunits is highlyconserved, therefore it is likely that the "deletion" in clone T95corresponds to splicing out of gustducin exon 6.

Another type of clone (T93, T85 and T77) from the cDNA library containedan insertion of 193 bp. Comparison with the sequence of the exon/intronboundaries of the genomic clone of murine transducin α subunit indicatesthat this insertion is due to the presence of an unspliced intronbetween exons 6 and 7.

PCR reactions using primers spanning exons 6 and 7 showed that theabberantly spliced (deleted) variant and the unspliced form are presentin taste-enriched cDNA at levels approximately one tenth that of thecorrectly spliced α gustducin cDNA. The amino acids present within exon6 (R₁₉₇ to N₂₄₁) are highly conserved for α_(i) subunits and transducinα subunits and have been implicated in guanine nucleotide binding. Ifthe deleted form of gustducin is actually produced it would differsignificantly in its guanine nucleotide binding properties and GTPaseactivities from other a proteins. The unspliced form of gustducin wouldproduce a truncated protein lacking the terminal 114 amino acids, whichwould also be altered in its guanine nucleotide binding properties andin its ability to interact with receptors if produced.

EXAMPLE 3

The gustducin-encoding cDNA from pSPORT-gustducin (see Example 1) wassubcloned into a protein fusion vector [pMal-C2, New England Biolabs(NEB), Beverly, Mass.]. To accomplish this construction, the HindIIIsite of pMal-C2 was converted to a NotI site and PCR performed usingclone T95 (see Example 2) as substrate was used to generate a ˜65 bplong 5' fragment of gustducin cDNA with a NaeI site at the 5' end and aHindIII site at the 3' end. These DNA sequences were ligated in athree-piece ligation to an ˜1530 bp piece of pSPORT-gustducin generatedby digestion of pSPORT-gustducin with HindIII and NotI. The resultingconstruct, which was designated pMal-C2-gustducin, encodes maltosebinding protein fused to gustducin. Cleavage of the fusion productproduces a 353 amino acid long gustducin product lacking only the aminoterminal methionine. Preliminary attempts to express pMal-C2-gustducinin E. coli using a maltose binding protein fusion and purificationsystem (NEB) resulted in a product which, when cleaved from maltosebinding protein, was immunologically reactive with gustducin specificantibody but did not have the expected GTP-binding or GTPase activity.

EXAMPLE 4

Expression of gustducin α subunit mRNA in various rat tissues wasassayed by Northern blot, primer extension and RNase protection.

Expression Products (mRNA) of the Gustducin α Subunit

Northern blot analysis of poly A+ mRNA from taste tissue using labeledgustducin α subunit DNA as a probe indicates three transcripts: aclosely spaced double ˜1700-1800 nt and a faint third band ˜1500 nt. Theproducts of primer extension reactions using cRNA (i.e., RNA generatedin vitro as run-off transcripts from the taste cell cDNA library) astemplate and gustducin specific primers indicated the same 5' terminusas indicated in FIG. 1. These results indicate that the full length αgustducin clone is ˜1700 nt in length as depicted in FIG. 1.

Tissue Expression of the Gustducin α Subunit

Tissue specific expression of gustducin α subunit transcripts wasassayed by RNase protection. The template RNAs used for RNase protectionwere total RNA or, in those cases in which abundant RNA was not readilyavailable, cDNA libraries were made from poly A+ mRNA, then cRNA wasmade from the libraries. RNase protection was done simultaneously withgustducin α subunit probes and actin probes to normalize for expression.All RNAse protection assays were done using a RNase protection kit(Ambion, Austin, Tex.) according to the method described in Krieg etal., Methods Enz., 155, 397415 (1987).

The RNase protection assays demonstrated the presence of gustducin αsubunit RNA only in taste tissue enriched preparations. No α subunit RNAwas detected in the non-taste lingual tissue, olfactory epithelium,retina, brain, liver, heart or kidney.

In situ hybridization using labeled gustducin α subunit RNA probesdemonstrated the presence of α gustducin mRNA in the taste buds ofcircumvallate, foliate, and fungiform papillae, tissues directlyinvolved in taste transduction. Gustducin mRNA was completely absentfrom lingual tissue not involved in taste transduction includingnon-sensory lingual epithelium, muscle, connective tissue and vonEbner's glands.

Gustducin α Subunit Expression Requires Afferent Innervation

To determine if the expression of gustducin α subunit mRNA is dependenton the presence of taste buds, in situ hybridizations using labeledgustducin -α subunit antisense RNA as probes were carried out on frozensections taken from rats whose tongues had been denervated. When thenerves innervating taste buds are severed, the buds degenerate and donot reappear unless the connections are restored. If gustducin α subunitmRNA is present only within the taste buds, it follows that upondegeneration of the taste buds, gustducin α subunit would no longer beexpressed.

In the rat, taste buds are innervated by branches of theglossopharyngeal, the facial, and the vagal cranial nerves. Theglossopharyngeal nerve innervates the circumvallate papilla, and sometaste buds of the foliate papillae. The chorda tympani innervates thefoliate papillae as well as the fungiform papillae of the anteriorportion of the tongue.

Two types of denervation were performed: (a) bilateral section of bothglossopharyngeal nerves and (b) unilateral section of the leftglossopharyngeal nerve and the left chorda tympani. The circumvallatepapilla is innervated only by the glossopharyngeal nerves; bilateralsectioning of these nerves causes the taste buds of this papilla todegenerate. Unilateral sectioning causes the taste buds of theipsilateral foliate papilla to degenerate, but leaves the taste buds ofthe contralateral foliate papilla intact. Fourteen days post-surgery (toallow full degeneration of taste buds) tissue sections containingfoliate and circumvallate papillae were subjected to in situhybridization with α gustducin anti-sense probe.

Following bilateral glossopharyngeal denervation the circumvallatepapilla was totally devoid of taste buds and gustducin α subunit mRNAexpression was likewise absent from the circumvallate papilla. Asexpected, taste buds expressing α gustducin mRNA were still present inthe foliate papillae of these rats (since input from the chorda tympaniremained). However, the number of taste buds in these papillae didappear to be reduced. Following unilateral sectioning of the left chordatympani and left glossopharyngeal nerve, the ipsilateral foliate papillawas devoid of taste buds and displayed no detectable expression ofgustducin α subunit mRNA, however, the contralateral foliate papillaretained taste buds which did express gustducin α subunit mRNA. Theseresults directly correlate the presence of innervated taste buds withgustducin α subunit expression.

EXAMPLE 5

Based on the amino acid sequence homology between the gustducin αsubunit and the transducin α subunits and on the taste cell specificexpression pattern of both the gustducin α subunit and the transducin αsubunit, it is reasonable to conclude that the roles of gustducin andtransducin in taste transduction is similar to the role of transducin inthe visual system. Gustducin and/or transducin are likely to transducetaste receptor activation into activation or inhibition of a taste celleffector such as cAMP or cGMP phosphodiesterase. Gustducin α subunitsand transducin α subunits may therefore be utilized in methods toidentify taste modifying agents that are capable of mimicking, blockingor inhibiting particular tastes. As indicated below, the specificidentification methods are designed by analogy to procedures employed tocharacterize activation and effector functions of known G proteins.

A first type of method identifies taste modifying agents that mimic orblock the effect of an activated taste receptor on the gustducin ortransducin α subunit. For example, one method contemplated by theinvention is analogous to an assay described in Cheung et al., FEBSLetters, 279(2), 277-280 (1991) wherein evidence of peptide activationof various G proteins was an increase in the rate of GTPγS binding by Gprotein α subunits. (GTPγS is a nonhydrolyzable form of GTP.) The methodtherefore may include the steps of incubating phospholipid vesicleshaving gustducin α subunit (bound to GDP) or transducin α subunit (boundto GDP) and G protein β and γ subunit (i.e., any purified β and γsubunits may be used) associated in biologically active form with aputative taste modifying agent and radioactively labeled GTPγS, anddetermining the rate of GTPγS binding by the α subunit in comparision toa standard rate (i.e., the rate of binding in the absence of the agent).An increase in the rate of binding indicates that the agent is a tastestimulator and a decrease in the rate of binding indicates that theagent is a taste inhibitor.

Another method of the first type is analogous to a different assaydescribed in Cheung et al., FEBS Letters, 279(2), 277-280 (1991) whereinevidence of peptide activation of various & proteins was an increase inthe rate of G protein α subunit GTPase activity. This method maytherefore comprise the steps of incubating phospholipid vesicles havinggustducin α subunit (bound to GDP) or transducin α subunit (bound toGDP) and G protein β and γ subunit associated in biologically activeform with a putative taste modifying agent and radioactively labeledGTP, and determining the rate of conversion of GTP to GDP by the αsubunit in comparison to the rate of conversion in the absence of theagent. An increase in the rate of conversion indicates that the agent isa taste stimulator and a decrease in the rate of conversion indicatesthat the agent is a taste inhibitor.

Yet another method of the first type contemplated by the invention isanalogous to an assay described in Konig et al., Proc. Natl. Acad Sci,USA, 86, 6878-6882 (1989) wherein evidence for transducin α subunitinteraction with an activated receptor (rhodopsin) is an increase inabsorbance at 380 nm. (It is likely that gustducin will interact withrhodopsin because the carboxyl terminal thirty-eight amino acids oftransducin [which have been shown to include the site of transducininteraction with rhodopsin, see Nishizuka et al., Eds., pp. 76-82 in TheBiology and Medicine of Signal Transduction, Raven Press, New York(1990)] are identical to the carboxyl terminal thirty-eight amino acidsof gustducin.) The method includes the steps of incubating washed diskmembranes having gustducin α subunit (bound to GDP) or transducin αsubunit (bound to GDP) associated with G protein β and γ subunits inbiologically active form with a putative taste modifying agent,subjecting the incubation mixture to photolyzing conditions (i.e., 532nm light), and determining absorption at 380 nm (vs. 417 nm) incomparison to absorption in the absence of the agent. An increase inabsorption at 380 nm indicates the agent is a taste stimulator and adecrease in absorption at 380 nm indicates that the agent is a tasteinhibitor.

A second type of method identifies taste modifying agents that mimic orblock the effect of activated gustducin or transducin α subunit (i.e.,subunit having bound GTP or GTPγS) on an effector. A contemplated methodof this type is analogous to assays described in Beavo-et al., Eds.,Chpt. 7 in Cyclic Nucleotide Phosphodiesterases: Structure, Regulationand Drug Action, John Wiley and Sons Ltd. (1990) and in Rarick et al.,Science, 256, 1031-1033 (1992), wherein phosphodiesterase (PDE)activation is evidence of transducin interaction with an effector, cGMPPDE. The method therefore may include the steps of incubating activatedgustducin α subunit or activated transducin α subunit with a putativetaste modifying agent and cAMP (or cGMP) PDE, and measuringphosphodiesterase activation by the α subunit in comparison to the levelof phosphodiesterase activity in the absence of the agent. An increasein activity indicates that the agent is a taste stimulator and adecrease in activity indicates that the agent is a taste inhibitor.

Peptides (e.g., fragments of antibodies to gustducin or transducin andpeptides corresponding to portions of gustducin or transducin) thatmimic or compete with a binding activity of the gustducin or transducinα subunits may be taste modifying agents. These peptides are likely toaffect the interaction of the gustducin/transducin α subunits withsensory receptors, cellular effectors and/or their associated β and γsubunits. See Rarick et al., supra, which describes a transducin αsubunit peptide that is capable of mimicking the activation of aphosphodiesterase by transducin. Examples of amino acid sequences ofsuch taste modifying peptides are: SEQ ID NOs: 1-3, which correspond tothe carboxyl terminal region of rat gustducin α subunit; SEQ ID NO: 4,which corresponds to the amino terminal portion of bovine transducin;SEQ ID NOs: 5-7, which correspond to the carboxyl terminal portion ofbovine transducin; SEQ ID NOs: 8-10, which correspond to loop peptidesof bovine rhodopsin; SEQ ID NO: 11 which corresponds to amino acids297-318 of rat gustducin; SEQ ID NO: 12 which corresponds to amino acids304-318 of rat gustducin; SEQ ID NO: 13 which corresponds to amino acids57-69 of rat gustducin; SEQ ID NO: 14 which corresponds to amino acids293-314 of bovine rod transducin; SEQ ID NO: 15 which corresponds toamino acids 300-314 of bovine rod transducin; SEQ ID NO: 16 whichcorresponds to amino acids 53-65 of bovine rod transducin; SEQ ID NO: 17which corresponds to amino acids 297-318 of bovine cone transducin; SEQID NO: 18 which corresponds to amino acids 304-318 of bovine conetransducin; and SEQ ID NO: 19 which corresponds to amino acids 57-69 ofbovine transducin.

EXAMPLE 6

Antibody substances (including monoclonal and polyclonal antibodies,chimeric and humanized antibodies, and antibody domains including Fab,Fab', F(ab')₂ and single chain domains, and Fv or single variabledomains) that are specific for the gustducin α subunit may be developedusing isolated natural or recombinant gustducin α subunit polypeptideproducts or host cells expressing such products on their surfaces. Theantibody substances may be utilized for blocking or inhibiting theligand/antiligand binding activities of gustducin as described in theforegoing paragraph and for purifying gustducin materials of theinvention.

The gustducin specific peptide YVNPRSREDQQLLLS (SEQ ID NO: 46)corresponding to amino acids 95-109 of the gustducin protein wassynthesized by Research Genetics (Huntsville, Ala.) on an eight-branchedchain lysine core [multiple antigen peptide, MAP, described in Tam,Proc. Natl. Acad. Sci. USA, 85: 5409-5413 (1988)] The MAP-peptide(denoted Gust-1) was used to inoculate rabbits to raise a polyclonalanti-peptide antiserum specific for this gustducin peptide. On day 0,preimmune sera was collected and then the popliteal lymph node wasinjected with the GUST-1 MAP (500 μg) in complete Freund's Adjuvant. Twoboosters, the first of 500 μg GUST-1 in incomplete Freund's adjuvant(IFA) and the second of 250 μg in IFA, were then injected intradermallyon days 14 and 42, respectively. Five ml immune serum was collected ondays 28 and 56. Subsequently, boosters of 100 μg of GUST-1 weresubcutaneously injected once a month. Immune serum was then collected 2weeks after each booster injection.

Western blot and immunohistochemistry experiments performed with thisgustducin specific antibody have demonstrated the presence of gustducinprotein in extracts from rat and bovine circumvallate and foliatepapillae. The gustducin specific antibody was also reactive with the E.coli maltose binding protein-gustducin fusion product of Example 3.

While the present invention has been described in terms of preferredembodiments, it is understood that variations and improvements willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations which come withinthe scope of the invention as claimed.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 46                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Glu Asp Lys Glu Ile Tyr Ser His Met Thr Cy - #s Ala Thr Asp Thr Gln      1               5   - #                10  - #                15               - - Asn Val                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Glu Asp Lys Glu Ile Tyr Ser His Met Thr Cy - #s Ala Thr Asp Thr Gln      1               5   - #                10  - #                15               - - Asn Val Lys                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Glu Asp Lys Glu Ile Tyr Ser His Met Thr Cy - #s Ala Thr Asp Thr Gln      1               5   - #                10  - #                15               - - Asn Val Lys Phe Val Phe Asp Ala Val Thr As - #p Ile Ile Ile Lys Glu                  20      - #            25      - #            30                   - - Asn Leu Lys Asp Cys Gly Leu Phe                                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Glu Glu Lys His Ser Arg Glu Leu Glu Lys Ly - #s Leu Lys Glu Asp Ala      1               5   - #                10  - #                15               - - Glu Lys Asp Ala Arg                                                                  20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Asp Val Lys Glu Ile Tyr Ser His Met Thr Cy - #s Ala Thr Asp Thr Gln      1               5   - #                10  - #                15               - - Asn Val                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Asp Val Lys Glu Ile Tyr Ser His Met Thr Cy - #s Ala Thr Asp Thr Gln      1               5   - #                10  - #                15               - - Asn Val Lys                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Ile Lys Glu Asn Leu Lys Asp Cys Gly Leu Ph - #e                          1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -  - -      (i) SEQUENCE CHARACTERISTICS:                                            (A) LENGTH: 13 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Lys Pro Met Ser Asn Phe Arg Phe Gly Glu As - #n His Ala                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Val Lys Glu Ala Ala Ala Gln Gln Gln Glu Se - #r Ala Thr Thr Gln Lys      1               5   - #                10  - #                15               - - Ala Glu Lys Glu Val Thr Arg                                                          20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Asn Lys Gln Phe Arg Asn Cys Met Val Thr Th - #r Leu                      1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Glu Asp Ala Gly Asn Tyr Ile Lys Asn Gln Ph - #e Leu Asp Leu Asn Leu      1               5   - #                10  - #                15               - - Lys Lys Glu Asp Lys Glu                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Lys Asn Gln Phe Leu Asp Leu Asn Leu Lys Ly - #s Glu Asp Lys Glu          1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - His Lys Asn Gly Tyr Ser Lys Gln Glu Cys Me - #t Glu Phe                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Glu Asp Ala Gly Asn Tyr Ile Lys Val Gln Ph - #e Leu Glu Leu Asn Met      1               5   - #                10  - #                15               - - Arg Arg Asp Val Lys Glu                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Lys Val Gln Phe Leu Glu Leu Asn Met Arg Ar - #g Asp Val Lys Glu                         5   - #                10  - #                15                - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - His Gln Asp Gly Tyr Ser Leu Glu Glu Cys Le - #u Glu Phe                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - Glu Asp Ala Gly Asn Tyr Ile Lys Ser Gln Ph - #e Leu Asp Leu Asn Met      1               5   - #                10  - #                15               - - Arg Lys Asp Val Lys Glu                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - Lys Ser Gln Phe Leu Asp Leu Asn Met Arg Ly - #s Asp Val Lys Glu          1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:19                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - His Gln Asp Gly Tyr Ser Pro Glu Glu Cys Le - #u Glu Tyr                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1703 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 114..1175                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - GACTGGTGCC TGCTGTTGGG AGCACTGCTC TGACGATCTA TCTCTAAACC AC -             #TGCTGTGC     60                                                                 - - TCTGTGTTTG AAAACTTTGA GCAAATCAAC TGCCCGTCCT CTAACAGCAA AA - #G ATG           116                                                                                        - #                  - #                  - #     Met                         - #                  - #                  - #       1        - - GGA AGT GGA ATT AGT TCA GAG AGC AAG GAG TC - #A GCC AAA AGG TCC AAA          164                                                                       Gly Ser Gly Ile Ser Ser Glu Ser Lys Glu Se - #r Ala Lys Arg Ser Lys                         5    - #              10    - #              15                  - - GAA CTG GAG AAG AAG CTT CAG GAA GAT GCT GA - #A CGA GAT GCA AGA ACT          212                                                                       Glu Leu Glu Lys Lys Leu Gln Glu Asp Ala Gl - #u Arg Asp Ala Arg Thr                    20         - #         25         - #         30                      - - GTG AAG TTG CTG CTA TTA GGA GCA GGT GAA TC - #T GGA AAA AGT ACT ATT          260                                                                       Val Lys Leu Leu Leu Leu Gly Ala Gly Glu Se - #r Gly Lys Ser Thr Ile                35             - #     40             - #     45                          - - GTT AAA CAA ATG AAG ATC ATC CAC AAG AAT GG - #T TAC AGT AAA CAA GAA          308                                                                       Val Lys Gln Met Lys Ile Ile His Lys Asn Gl - #y Tyr Ser Lys Gln Glu            50                 - # 55                 - # 60                 - # 65       - - TGC ATG GAG TTT AAA GCA GTG GTT TAC AGT AA - #C ACG TTG CAG TCC ATC          356                                                                       Cys Met Glu Phe Lys Ala Val Val Tyr Ser As - #n Thr Leu Gln Ser Ile                            70 - #                 75 - #                 80              - - CTG GCC ATT GTG AAA GCC ATG ACT ACA CTA GG - #G ATT GAT TAT GTC AAT          404                                                                       Leu Ala Ile Val Lys Ala Met Thr Thr Leu Gl - #y Ile Asp Tyr Val Asn                        85     - #             90     - #             95                  - - CCG AGA AGT AGA GAG GAC CAA CAA CTG CTT CT - #C TCC ATG GCA AAC ACA          452                                                                       Pro Arg Ser Arg Glu Asp Gln Gln Leu Leu Le - #u Ser Met Ala Asn Thr                   100          - #       105          - #       110                      - - CTA GAA GAT GGT GAC ATG ACG CCT CAG TTG GC - #T GAA ATA ATT AAA CGT          500                                                                       Leu Glu Asp Gly Asp Met Thr Pro Gln Leu Al - #a Glu Ile Ile Lys Arg               115              - #   120              - #   125                          - - CTG TGG GGC GAT CCA GGA ATT CAA GCC TGC TT - #C GAA AGG GCA TCT GAA          548                                                                       Leu Trp Gly Asp Pro Gly Ile Gln Ala Cys Ph - #e Glu Arg Ala Ser Glu           130                 1 - #35                 1 - #40                 1 -      #45                                                                              - - TAC CAG CTC AAT GAC TCT GCA GCT TAC TAC CT - #T AAT GAC TTA GAT        AGA      596                                                                    Tyr Gln Leu Asn Asp Ser Ala Ala Tyr Tyr Le - #u Asn Asp Leu Asp Arg                          150  - #               155  - #               160              - - CTC ACA GCC CCT GGG TAT GTG CCA AAT GAA CA - #A GAC GTT CTA CAT TCC          644                                                                       Leu Thr Ala Pro Gly Tyr Val Pro Asn Glu Gl - #n Asp Val Leu His Ser                       165      - #           170      - #           175                  - - CGG GTG AAA ACC ACT GGT ATC ATT GAA ACT CA - #A TTC TCC TTT AAA GAC          692                                                                       Arg Val Lys Thr Thr Gly Ile Ile Glu Thr Gl - #n Phe Ser Phe Lys Asp                   180          - #       185          - #       190                      - - TTG AAC TTC AGA ATG TTT GAT GTA GGT GGC CA - #G AGA TCA GAA AGA AAG          740                                                                       Leu Asn Phe Arg Met Phe Asp Val Gly Gly Gl - #n Arg Ser Glu Arg Lys               195              - #   200              - #   205                          - - AAA TGG ATC CAC TGC TTT GAA GGA GTG ACG TG - #C ATT ATA TTT TGT GCA          788                                                                       Lys Trp Ile His Cys Phe Glu Gly Val Thr Cy - #s Ile Ile Phe Cys Ala           210                 2 - #15                 2 - #20                 2 -      #25                                                                              - - GCC CTA AGT GCC TAC GAC ATG GTA CTT GTA GA - #A GAT GAA GAG GTG        AAC      836                                                                    Ala Leu Ser Ala Tyr Asp Met Val Leu Val Gl - #u Asp Glu Glu Val Asn                          230  - #               235  - #               240              - - AGA ATG CAT GAA AGT CTT CAC CTC TTC AAC AG - #C ATC TGT AAT CAC AAG          884                                                                       Arg Met His Glu Ser Leu His Leu Phe Asn Se - #r Ile Cys Asn His Lys                       245      - #           250      - #           255                  - - TAT TTT GCA ACC ACC TCC ATT GTT CTG TTT CT - #T AAC AAG AAA GAT CTC          932                                                                       Tyr Phe Ala Thr Thr Ser Ile Val Leu Phe Le - #u Asn Lys Lys Asp Leu                   260          - #       265          - #       270                      - - TTC CAG GAG AAA GTG ACC AAG GTG CAC CTC AG - #C ATC TGT TTC CCA GAA          980                                                                       Phe Gln Glu Lys Val Thr Lys Val His Leu Se - #r Ile Cys Phe Pro Glu               275              - #   280              - #   285                          - - TAC ACT GGA CCA AAT ACA TTC GAA GAT GCA GG - #G AAC TAC ATC AAG AAC         1028                                                                       Tyr Thr Gly Pro Asn Thr Phe Glu Asp Ala Gl - #y Asn Tyr Ile Lys Asn           290                 2 - #95                 3 - #00                 3 -      #05                                                                              - - CAG TTC CTA GAC CTG AAC TTA AAA AAA GAA GA - #T AAG GAA ATC TAT        TCT     1076                                                                    Gln Phe Leu Asp Leu Asn Leu Lys Lys Glu As - #p Lys Glu Ile Tyr Ser                          310  - #               315  - #               320              - - CAC ATG ACC TGC GCT ACT GAC ACA CAA AAC GT - #C AAA TTC GTG TTT GAT         1124                                                                       His Met Thr Cys Ala Thr Asp Thr Gln Asn Va - #l Lys Phe Val Phe Asp                       325      - #           330      - #           335                  - - GCC GTG ACA GAT ATA ATA ATA AAA GAG AAC CT - #C AAA GAC TGT GGG CTC         1172                                                                       Ala Val Thr Asp Ile Ile Ile Lys Glu Asn Le - #u Lys Asp Cys Gly Leu                   340          - #       345          - #       350                      - - TTC TGAGCAACCT GTTTGCTACC ACTTGTGATG GCTATAGTCT TTTTAAGAC - #A              1225                                                                       Phe                                                                            - - TAAAAAGGTG CTGTGTATTA GCTTGGATAG ATATTAACTG ATTTAGAAAT GT -             #GACTAGCA   1285                                                                 - - TTATAAAACA AAAAAATTCA CACAAAAATA TTACTGTGAT ATCACGTATA TC -            #TGGGTACG   1345                                                                 - - GTTTTCTTGG GGAATGGAGG GTAGAGTTGC TGATGTTCTA AATCTGAAAT CT -            #GATGTATC   1405                                                                 - - TGGTAACTGT CACAATATAC ATTCATGCTA CTAAAGTTTT TTGGAAGTGA GC -            #TGTAAGTG   1465                                                                 - - ACCAATTTTT AATCATAGAG TAAACCTCAG AATGTGCTAT AACATTGCCC CA -            #GCTAGATT   1525                                                                 - - TTGAAAGCAT TCAAAGTCAT GTCTGTACTA CAGAAACTGT ACAAAATGAA CA -            #AGGTATAA   1585                                                                 - - TTTTGGTCAT CAGCCTTTCA ATTAGGCTGC CACAAGCACA CACAGTACAT GC -            #TTTTATTG   1645                                                                 - - ATGGGAAATT GTATGTGTAA AATAAATATA TATATAATAA AAAAAAAAAA AA -            #AAAAAA     1703                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 354 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - Met Gly Ser Gly Ile Ser Ser Glu Ser Lys Gl - #u Ser Ala Lys Arg        Ser                                                                               1               5 - #                 10 - #                 15             - - Lys Glu Leu Glu Lys Lys Leu Gln Glu Asp Al - #a Glu Arg Asp Ala Arg                   20     - #             25     - #             30                  - - Thr Val Lys Leu Leu Leu Leu Gly Ala Gly Gl - #u Ser Gly Lys Ser Thr               35         - #         40         - #         45                      - - Ile Val Lys Gln Met Lys Ile Ile His Lys As - #n Gly Tyr Ser Lys Gln           50             - #     55             - #     60                          - - Glu Cys Met Glu Phe Lys Ala Val Val Tyr Se - #r Asn Thr Leu Gln Ser       65                 - # 70                 - # 75                 - # 80       - - Ile Leu Ala Ile Val Lys Ala Met Thr Thr Le - #u Gly Ile Asp Tyr Val                       85 - #                 90 - #                 95              - - Asn Pro Arg Ser Arg Glu Asp Gln Gln Leu Le - #u Leu Ser Met Ala Asn                  100      - #           105      - #           110                  - - Thr Leu Glu Asp Gly Asp Met Thr Pro Gln Le - #u Ala Glu Ile Ile Lys              115          - #       120          - #       125                      - - Arg Leu Trp Gly Asp Pro Gly Ile Gln Ala Cy - #s Phe Glu Arg Ala Ser          130              - #   135              - #   140                          - - Glu Tyr Gln Leu Asn Asp Ser Ala Ala Tyr Ty - #r Leu Asn Asp Leu Asp      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Arg Leu Thr Ala Pro Gly Tyr Val Pro Asn Gl - #u Gln Asp Val Leu        His                                                                                             165  - #               170  - #               175             - - Ser Arg Val Lys Thr Thr Gly Ile Ile Glu Th - #r Gln Phe Ser Phe Lys                  180      - #           185      - #           190                  - - Asp Leu Asn Phe Arg Met Phe Asp Val Gly Gl - #y Gln Arg Ser Glu Arg              195          - #       200          - #       205                      - - Lys Lys Trp Ile His Cys Phe Glu Gly Val Th - #r Cys Ile Ile Phe Cys          210              - #   215              - #   220                          - - Ala Ala Leu Ser Ala Tyr Asp Met Val Leu Va - #l Glu Asp Glu Glu Val      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asn Arg Met His Glu Ser Leu His Leu Phe As - #n Ser Ile Cys Asn        His                                                                                             245  - #               250  - #               255             - - Lys Tyr Phe Ala Thr Thr Ser Ile Val Leu Ph - #e Leu Asn Lys Lys Asp                  260      - #           265      - #           270                  - - Leu Phe Gln Glu Lys Val Thr Lys Val His Le - #u Ser Ile Cys Phe Pro              275          - #       280          - #       285                      - - Glu Tyr Thr Gly Pro Asn Thr Phe Glu Asp Al - #a Gly Asn Tyr Ile Lys          290              - #   295              - #   300                          - - Asn Gln Phe Leu Asp Leu Asn Leu Lys Lys Gl - #u Asp Lys Glu Ile Tyr      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ser His Met Thr Cys Ala Thr Asp Thr Gln As - #n Val Lys Phe Val        Phe                                                                                             325  - #               330  - #               335             - - Asp Ala Val Thr Asp Ile Ile Ile Lys Glu As - #n Leu Lys Asp Cys Gly                  340      - #           345      - #           350                  - - Leu Phe                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 354 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - Met Gly Ser Gly Ala Ser Ala Glu Asp Lys Gl - #u Leu Ala Lys Arg Ser      1               5   - #                10  - #                15               - - Lys Glu Leu Glu Lys Lys Leu Gln Glu Asp Al - #a Asp Lys Glu Ala Lys                  20      - #            25      - #            30                   - - Thr Val Lys Leu Leu Leu Leu Gly Ala Gly Gl - #u Ser Gly Lys Ser Thr              35          - #        40          - #        45                       - - Ile Val Lys Gln Met Lys Ile Ile His Gln As - #p Gly Tyr Ser Pro Glu          50              - #    55              - #    60                           - - Glu Cys Leu Glu Tyr Lys Ala Ile Ile Tyr Gl - #y Asn Val Leu Gln Ser      65                  - #70                  - #75                  - #80        - - Ile Leu Ala Ile Ile Arg Ala Met Pro Thr Le - #u Gly Ile Asp Tyr Ala                      85  - #                90  - #                95               - - Glu Val Ser Cys Val Asp Asn Gly Arg Gln Le - #u Asn Asn Leu Ala Asp                  100      - #           105      - #           110                  - - Ser Ile Glu Glu Gly Thr Met Pro Pro Glu Le - #u Val Glu Val Ile Arg              115          - #       120          - #       125                      - - Lys Leu Trp Lys Asp Gly Gly Val Gln Ala Cy - #s Phe Asp Arg Ala Ala          130              - #   135              - #   140                          - - Glu Tyr Gln Leu Asn Asp Ser Ala Ser Tyr Ty - #r Leu Asn Gln Leu Asp      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Arg Ile Thr Ala Pro Asp Tyr Leu Pro Asn Gl - #u Gln Asp Val Leu        Arg                                                                                             165  - #               170  - #               175             - - Ser Arg Val Lys Thr Thr Gly Ile Ile Glu Th - #r Lys Phe Ser Val Lys                  180      - #           185      - #           190                  - - Asp Leu Asn Phe Arg Met Phe Asp Val Gly Gl - #y Gln Arg Ser Glu Arg              195          - #       200          - #       205                      - - Lys Lys Trp Ile His Cys Phe Glu Gly Val Th - #r Cys Ile Ile Phe Cys          210              - #   215              - #   220                          - - Ala Ala Leu Ser Ala Tyr Asp Met Val Leu Va - #l Glu Asp Asp Glu Val      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asn Arg Met His Glu Ser Leu His Leu Phe As - #n Ser Ile Cys Asn        His                                                                                             245  - #               250  - #               255             - - Lys Phe Phe Ala Ala Thr Ser Ile Val Leu Ph - #e Leu Asn Lys Lys Asp                  260      - #           265      - #           270                  - - Leu Phe Glu Glu Lys Ile Lys Lys Val His Le - #u Ser Ile Cys Phe Pro              275          - #       280          - #       285                      - - Glu Tyr Asp Gly Asn Asn Ser Tyr Glu Asp Al - #a Gly Asn Tyr Ile Lys          290              - #   295              - #   300                          - - Ser Gln Phe Leu Asp Leu Asn Met Arg Lys As - #p Val Lys Glu Ile Tyr      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ser His Met Thr Cys Ala Thr Asp Thr Gln As - #n Val Lys Phe Val        Phe                                                                                             325  - #               330  - #               335             - - Asp Ala Val Thr Asp Ile Ile Ile Lys Glu As - #n Leu Lys Asp Cys Gly                  340      - #           345      - #           350                  - - Leu Phe                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 350 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - Met Gly Ala Gly Ala Ser Ala Glu Glu Lys Hi - #s Ser Arg Glu Leu Glu      1               5   - #                10  - #                15               - - Lys Lys Leu Lys Glu Asp Ala Glu Lys Asp Al - #a Arg Thr Val Lys Leu                  20      - #            25      - #            30                   - - Leu Leu Leu Gly Ala Gly Glu Ser Gly Lys Se - #r Thr Ile Val Lys Gln              35          - #        40          - #        45                       - - Met Lys Ile Ile His Gln Asp Gly Tyr Ser Le - #u Glu Glu Cys Leu Glu          50              - #    55              - #    60                           - - Phe Ile Ala Ile Ile Tyr Gly Asn Thr Leu Gl - #n Ser Ile Leu Ala Ile      65                  - #70                  - #75                  - #80        - - Val Arg Ala Met Thr Thr Leu Asn Ile Gln Ty - #r Gly Asp Ser Ala Arg                      85  - #                90  - #                95               - - Gln Asp Asp Ala Arg Lys Leu Met His Met Al - #a Asp Thr Ile Glu Glu                  100      - #           105      - #           110                  - - Gly Thr Met Pro Lys Glu Met Ser Asp Ile Il - #e Gln Arg Leu Trp Lys              115          - #       120          - #       125                      - - Asp Ser Gly Ile Gln Ala Cys Phe Asp Arg Al - #a Ser Glu Tyr Gln Leu          130              - #   135              - #   140                          - - Asn Asp Ser Ala Gly Tyr Tyr Leu Ser Asp Le - #u Glu Arg Leu Val Thr      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Pro Gly Tyr Val Pro Thr Glu Gln Asp Val Le - #u Arg Ser Arg Val        Lys                                                                                             165  - #               170  - #               175             - - Thr Thr Gly Ile Ile Glu Thr Gln Phe Ser Ph - #e Lys Asp Leu Asn Phe                  180      - #           185      - #           190                  - - Arg Met Phe Asp Val Gly Gly Gln Arg Ser Gl - #u Arg Lys Lys Trp Ile              195          - #       200          - #       205                      - - His Cys Phe Glu Gly Val Thr Cys Ile Ile Ph - #e Ile Ala Ala Leu Ser          210              - #   215              - #   220                          - - Ala Tyr Asp Met Val Leu Val Glu Asp Asp Gl - #u Val Asn Arg Met His      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Glu Ser Leu His Leu Phe Asn Ser Ile Cys As - #n His Arg Tyr Phe        Ala                                                                                             245  - #               250  - #               255             - - Thr Thr Ser Ile Val Leu Phe Leu Asn Lys Ly - #s Asp Val Phe Ser Glu                  260      - #           265      - #           270                  - - Lys Ile Lys Lys Ala His Leu Ser Ile Cys Ph - #e Pro Asp Tyr Asn Gly              275          - #       280          - #       285                      - - Pro Asn Thr Tyr Glu Asp Ala Gly Asn Tyr Il - #e Lys Val Gln Phe Leu          290              - #   295              - #   300                          - - Glu Leu Asn Met Arg Arg Asp Val Lys Glu Il - #e Tyr Ser His Met Thr      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Cys Ala Thr Asp Thr Gln Asn Val Lys Phe Va - #l Phe Asp Ala Val        Thr                                                                                             325  - #               330  - #               335             - - Asp Ile Ile Ile Lys Glu Asn Leu Lys Asp Cy - #s Gly Leu Phe                          340      - #           345      - #           350                  - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 354 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #/note= "Positions indicated as Xaa                       represent - #nonconserved amino acids."                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - Met Gly Ser Gly Ala Ser Ala Glu Xaa Lys Gl - #u Xaa Ala Lys Arg Ser      1               5   - #                10  - #                15               - - Lys Glu Leu Glu Lys Lys Leu Gln Glu Asp Al - #a Glu Lys Asp Ala Arg                  20      - #            25      - #            30                   - - Thr Val Lys Leu Leu Leu Leu Gly Ala Gly Gl - #u Ser Gly Lys Ser Thr              35          - #        40          - #        45                       - - Ile Val Lys Gln Met Lys Ile Ile His Gln As - #p Gly Tyr Ser Xaa Glu          50              - #    55              - #    60                           - - Glu Cys Leu Glu Phe Lys Ala Ile Ile Tyr Gl - #y Asn Thr Leu Gln Ser      65                  - #70                  - #75                  - #80        - - Ile Leu Ala Ile Val Arg Ala Met Thr Thr Le - #u Gly Ile Asp Tyr Xaa                      85  - #                90  - #                95               - - Xaa Xaa Xaa Xaa Xaa Asp Asp Xaa Arg Xaa Le - #u Xaa Xaa Met Ala Asp                  100      - #           105      - #           110                  - - Thr Ile Glu Glu Gly Thr Met Pro Pro Glu Le - #u Xaa Glu Ile Ile Xaa              115          - #       120          - #       125                      - - Arg Leu Trp Lys Asp Xaa Gly Ile Gln Ala Cy - #s Phe Asp Arg Ala Ser          130              - #   135              - #   140                          - - Glu Tyr Gln Leu Asn Asp Ser Ala Xaa Tyr Ty - #r Leu Asn Asp Leu Asp      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Arg Leu Thr Ala Pro Gly Tyr Val Pro Asn Gl - #u Gln Asp Val Leu        Arg                                                                                             165  - #               170  - #               175             - - Ser Arg Val Lys Thr Thr Gly Ile Ile Glu Th - #r Gln Phe Ser Phe Lys                  180      - #           185      - #           190                  - - Asp Leu Asn Phe Arg Met Phe Asp Val Gly Gl - #y Gln Arg Ser Glu Arg              195          - #       200          - #       205                      - - Lys Lys Trp Ile His Cys Phe Glu Gly Val Th - #r Cys Ile Ile Phe Cys          210              - #   215              - #   220                          - - Ala Ala Leu Ser Ala Tyr Asp Met Val Leu Va - #l Glu Asp Asp Glu Val      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asn Arg Met His Glu Ser Leu His Leu Phe As - #n Ser Ile Cys Asn        His                                                                                             245  - #               250  - #               255             - - Lys Tyr Phe Ala Thr Thr Ser Ile Val Leu Ph - #e Leu Asn Lys Lys Asp                  260      - #           265      - #           270                  - - Leu Phe Xaa Glu Lys Ile Lys Lys Val His Le - #u Ser Ile Cys Phe Pro              275          - #       280          - #       285                      - - Glu Tyr Xaa Gly Pro Asn Thr Tyr Glu Asp Al - #a Gly Asn Tyr Ile Lys          290              - #   295              - #   300                          - - Asp Ala Val Thr Asp Ile Ile Ile Lys Glu As - #n Leu Lys Asp Cys Gly                        - #  340               - #  345               - #  350       - - Leu Phe                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - Lys Trp Ile His Cys Phe                                                  1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - CGGATCCAAR TGGATHCAYT GYTT          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - - Phe Leu Asn Lys Lys Asp                                                  1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - - GGAATTCRTC YTTYTTRTTN AGRAA          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                              - - GGAATTCRTC YTTYTTRTTY AARAA          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                              - - Asp Val Gly Gly Gln Arg                                                  1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                              - - GTCTAGAGAY GTNGGNGGNC ARMG          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                              - - Val Phe Asp Ala Val Thr Asp                                              1               5                                                              - - (2) INFORMATION FOR SEQ ID NO:33:                                         - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                              - - CCGAATTCTC NGTNACNGCR TCRAANAC         - #                  - #                 28                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34                               - - Thr Ile Val Lys Gln Met                                                   1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -      (ii) MOLECULE TYPE: DNA                                              - -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #35:                         - - CCGAATTCAC NATNGTNAAR CARATG          - #                  - #                  26                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                              - - Phe Leu Asn Lys Gln Asp                                                   1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                              - - CCGAATTCRT CYTGYTTRTT NARRAA          - #                  - #                  26                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:38:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                              - - His Leu Phe Asn Ser Ile Cys                                               1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:39:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                              - - CCGGATCCGC ACCTGTTCAA CAGCATCT         - #                  - #                 28                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:40:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #40:                         - - Lys Tyr Phe Ala Thr Thr Ser                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:41:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                              - - CCGGATCCGA GGTGGTTGCA AAATACTT         - #                  - #                 28                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:42:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                              - - Leu Ala Glu Ile Ile Lys Arg                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:43:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                              - - CGGATCCGAC GTTTAATTAT TTCAGCCAA         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:44:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                              - - TCGACCCACG CGTCCG             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:45:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                              - - GGGCGGCCGC                - #                  - #                      - #       110                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:46                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46                               - - Tyr Val Asn Pro Arg Ser Arg Glu Asp Gln Gl - #n Leu Leu Leu Ser          1               5   - #                10  - #                15             __________________________________________________________________________

I claim:
 1. A method for identifying a taste modifying agent comprisingthe steps of incubating phospholipid vesicles having gustducin α subunitor transducin α subunit and G protein β and γ subunits associated inbiologically active form with said agent and GTPγS, and determining therate of GTPγS binding by said α subunit in comparison to a standard ratewherein an increase in the rate of binding indicates that the agent is ataste stimulator and a decrease in the rate of binding indicates thatthe agent is a taste inhibitor.
 2. A method for identifying a tastemodifying agent comprising the steps incubating phospholipid vesicleshaving gustducin α subunit or transducin α subunit and G protein β and γsubunits associated in biologically active form with said agent and GTP,and determining the rate of conversion of GTP to GDP by said α subunitin comparison to a standard rate wherein an increase in the rate ofconversion indicates that the agent is a taste stimulator and a decreasein the rate of conversion indicates that the agent is a taste inhibitor.